Figure 5.

Carfilzomib combined with an anti-VEGFR2 antibody inhibits HCC progression. (A) Kaplan–Meier survival analysis with the log-rank test of NOD/SCID mice subcutaneously xenotransplanted with Huh7 cells. The mice were treated with control IgG and vehicle (red), control IgG and carfilzomib (orange), anti-VEGFR2 antibody and vehicle (green), or anti-VEGFR2 antibody and carfilzomib (blue) (n = 7 in each group). The control vehicle (200 μL/day) or carfilzomib (6 mg/kg/day) was injected intraperitoneally on days 1, 2, 8, and 9, as indicated by upper black arrows. Control IgG (40 mg/kg) or anti-mouse VEGFR2 monoclonal antibodies (DC101, 40 mg/kg) were injected intraperitoneally on days 1, 4, 8, and 11, as indicated by lower black arrows. (B) Photomicrographs of subcutaneous tumors that developed in NOD/SCID mice treated with the indicated reagents (n = 4 in each group). Mice were euthanized on day 14. Comparison of tumors with phosphate-buffered saline (control), carfilzomib (6 mg/kg), DC101 (40 mg/kg), or combined treatment with carfilzomib and DC101 for 2 weeks. (C) Subcutaneous tumor volume curves of Huh7 cells treated with the indicated reagents (n = 4 in each group, means ± SEM). (D) Subcutaneous tumor volume (upper panel) and weight (lower panel) in NOD/SCID mice treated with the indicated reagents on day 14 (one-way ANOVA test). (E) Representative immunohistochemistry staining images of FOXM1, AFP, and Ki-67 in subcutaneous tumors from NOD/SCID mice treated with the indicated reagents (scale bar = 100 μm). (F) Percentages of AFP-positive (left panel), FOXM1-positive (middle panel), and Ki-67-positive cells (right panel) counted three times from four independent immunohistochemistry staining images of subcutaneous tumors from NOD/SCID mice treated with the indicated reagents (means ± SEM, one-way ANOVA test). (G) Western blot of FOXM1 and β-actin in subcutaneous tumors from NOD/SCID mice treated with the indicated reagents.